The dimorphic fungus Histoplasma capsulatum (Hc) is a facultative intracellular pathogen of macrophages that causes respiratory and systemic disease. The essential nutrient iron lies at the competitive interface between the mammalian host and nearly all microbial pathogens, including Hc. The host displays both constitutive and inducible iron sequestration mechanisms. Iron limitation acts as an important host defense mechanism against Hc in human and mouse macrophage cell culture infection models. As a successful pathogen, Hc expresses iron acquisition mechanisms to obtain this nutrient in the competitive host environment in which it resides during infection. Several Hc iron acquisition mechanisms have been demonstrated or proposed. Hc produces extracellular hydroxamate siderophores during iron-limited growth in vitro. Siderophores may compete with host iron-binding compounds such as transferrin (Tf) to acquire iron for the fungus. Hc also practices ferric reduction via at least three moieties - an extracellular ferric reductase enzyme, extracellular ferric reductant(s), and cell-surface ferric reducing agent(s). Reduction of ferric to ferrous iron causes removal from both host (e.g., Tf) and fungal (siderophore) iron-binding compounds. Finally, Tf releases iron at sufficiently acidic pH, to which Hc may be exposed in its macrophage intracellular compartment. Our hypothesis is that since iron acquisition is essential to Hc's pathogenesis, one or more or a combination of the mechanisms for accomplishing this process may provide effective new antifungal vaccine components and/or antibiotic targets. We propose to examine and determine pathogenic relevance of iron acquisition components and mechanisms for this fungus in the following three Specific Aims. 1) Identify and characterize physiologically relevant iron sources and processes for iron utilization in vitro and during intracellular infection using biochemical approaches. 2) Purify the Hc extracellular ferric reductase, clone the encoding gene, and examine its expression under pathogenically relevant conditions. 3) Identify other Hc structural, biosynthetic, and regulatory genes involved in iron acquisition by molecular genetic approaches and determine their importance in Hc intracellular infection.